Intermediates and methods for synthesizing calicheamicin derivatives

ABSTRACT

The present invention relates to intermediates of Formula I 
     
       
         
         
             
             
         
       
     
     and to methods of synthesizing and purifying calicheamicin derivatives.

FIELD OF THE INVENTION

The invention relates to synthesis of calicheamicin derivatives. Theinvention also relates to intermediates and linker molecules useful forpreparing calicheamicin derivatives and for conjugating a calicheamicinto a biomacromolecule such as a monoclonal antibody.

BACKGROUND OF THE INVENTION

MYLOTARG® (gemtuzumab ozogamicin) consists of a monoclonal antibodyagainst CD33 that is bound to calicheamicin by means of anacid-hydrolyzable linker. The commercial product was marketed as thefirst antibody-targeted chemotherapeutic agent and was approved for thetreatment of acute myeloid leukemia (AML) in elderly patients.Inotuzumab ozogamicin is a CD22 antibody linked to a calicheamicincurrently in clinical trials for treatment of certain types of cancer.

The potent family of antibacterial and antitumor agents knowncollectively as the calicheamicins, or the LL-E33288 complex, aredisclosed in U.S. Pat. No. 4,970,198. U.S. Pat. No. 5,053,394 alsodiscloses methyltrisulfide antibacterial and antitumor agents. Thesecompounds in U.S. Pat. No. 4,970,198 and U.S. Pat. No. 5,053,394 containa methyltrisulfide group that can be reacted with appropriate thiols toform disulfides while at the same time introducing a functional groupsuch as a hydrazide or similar nucleophile. Examples of this reactionwith the calicheamicins are given in U.S. Pat. No. 5,053,394. U.S. Pat.No. 5,770,70 is directed to a process for preparing targeted forms ofdisulfide compounds of the LL-E33288 complex. A linker,4-(4-acetyl-phenoxy)butanoic acid, is condensed with an N-acetyl gammacalicheamicin dimethyl hydrazide compound to afford the carboxylicacid-hydrazone which is further treated with N-hydroxysuccinimide togive the OSu ester (N-succinimidyloxy) which is ready for conjugationwith a chosen biomacromolecule.

U.S. Pat. No. 8,273,862 describes a synthetic method for constructinglinker intermediate molecules (termed “trilinker-activated esters” or“trifunctional linker intermediates” therein). These linkerintermediates can be conjugated to calicheamicins to preparecalicheamicin derivatives that can then be further conjugated tobiomacromolecules such as monoclonal antibodies. In the steps leading tothe preparation of the linker intermediate, the synthetic methoddescribed in U.S. Pat. No. 8,273,862 uses a mercapto compound (“compound2” therein), for example 3-methyl-3-mercaptobutanoic acid hydrazide, asan intermediate.

WO 2008/147765 describes a method for synthesizing themercapto-containing intermediates, such as 3-methyl-3-mercaptobutanoicacid hydrazide, that are useful in preparing linker intermediates andcalicheamicin derivatives as described in the preceding paragraph. WO2008/147765 alternatively refers to the 3-methyl-3-mercaptobutanoic acidhydrazide as the “DMH linker”. WO 2008/147765 notes that3-methyl-3-mercaptobutanoic acid hydrazide is a preferredmercapto-containing N-acylhydrazine for the purpose of linkingcalicheamicin to monoclonal antibodies to make, for instance, gemtuzumabozogamicin or inotuzumab ozogamicin.

In WO 2008/147765, 3-methyl-3-mercaptobutanoic acid hydrazide isprepared by removing the benzyl protecting group from the compoundp-methoxybenzylthioether hydrazide under acidic conditions. In order toobtain p-methoxybenzylthioether hydrazide, WO 2008/137765 teaches firstreacting p-methoxybenzylthioether acid with oxalyl chloride in methylenechloride to form p-methoxybenzylthioether acid chloride. Thep-methoxybenzylthioether acid chloride is then added to a mixture ofanhydrous hydrazine and methylene chloride to obtain thep-methoxybenzylthioether hydrazide. However, as is described in WO2008/147765, the two reactants p-methyloxybenzylthioether acid andp-methoxybenzylthioether acid chloride themselves together generate anundesired by-product, bis-methoxybenzylthioether hydrazide, resulting inlower yield and quality. Furthermore, as described in WO 2008/147765,due to the reactive and unstable nature of the acid chloride moleculep-methoxybenzylthioether acid chloride, anhydrous hydrazine and lowtemperatures, e.g. −70° C., must be employed.

Another aspect in the preparation of calicheamicin derivatives involveschemically connecting the calicheamicin to the linker molecule. In orderto conjugate the calicheamicin to the linker intermediate in U.S. Pat.No. 8,273,862, a final reaction between a calicheamicin and a “trilinkeractivated ester” (or “trifunctional linker intermediate”) is conducted.Structurally, calicheamicins contain a trisulfide moiety, as explainedabove, that is used in their derivatization, and the chemistry of thisreaction between the trisulfide moiety of the calicheamicin and thetrilinker activated ester is important in realizing good yields andpurity. Previously used sulfur exchange reactions for calicheamicinderivatives have given complex reaction mixtures, multiple byproducts,and low yields.

Finally, the calicheamicin derivative is to be purified after itsformation. The process of U.S. Pat. No. 8,273,862 involves purificationof the calicheamicin derivative comprising a normal phase chromatographystep. The normal phase chromatography step in the purification processdescribed in U.S. Pat. No. 8,273,862 uses methylene chloride as asolvent, and, as stated above, exposure to methylene chloride abovevarious minimum levels is considered to pose potential health hazards.When using normal phase chromatography including methylene chloride,precautions to limit exposure to methylene chloride must therefore betaken.

SUMMARY OF THE INVENTION

The present invention provides new intermediates and methods forsynthesizing and purifying linker intermediates that are useful forconjugating calicheamicin antitumor antibiotics to biomacromolecules,such as monoclonal antibodies. The present invention further providesnew methods for synthesizing a calicheamicin derivative, whichderivative comprises a calicheamicin covalently bonded to a linker. Acalicheamicin derivative so prepared can be conjugated to abiomacromolecule, such as a monoclonal antibody to make an antibody-drugconjugate. The intermediates and the synthetic methods of the presentinvention can for example be used to prepare a calicheamicin derivativefor manufacturing gemtuzumab ozogamicin (MYLOTARG®) or inotuzumabozogamicin.

The subject invention provides improvements to the prior processes forsynthesizing and purifying calicheamicin derivatives that overcome someof the problems associated with these prior processes.

As explained above, WO 2008/137765 teaches first reacting anintermediate p-methoxybenzylthioether acid with oxalyl chloride inmethylene chloride to form a p-methoxybenzylthioether acid chlorideintermediate useful for synthesizing a calicheamicin derivative. In WO2008/137765, the p-methoxybenzylthioether acid chloride intermediate isthen added to a mixture of anhydrous hydrazine and methylene chloride toobtain a p-methoxybenzylthioether hydrazide intermediate. However, as isdescribed in WO 2008/147765, the two reactantsp-methyloxybenzylthioether acid and p-methoxybenzylthioether acidchloride themselves together generate an undesired by-product,bis-methoxybenzylthioether hydrazide, resulting in lower yield andquality. The present invention solves this problem of thebis-methoxybenzylthioether hydrazide by-product altogether by avoidingcompletely the use of p-methoxybenzylthioether acid chloride as anintermediate. By avoiding completely the acid chloride intermediatep-methoxybenzylthioether acid chloride, the subject inventionfurthermore now permits use of hydrated forms of hydrazine that do notrequire the same special handling procedures as does anhydrous hydrazineand the new method also avoids the cumbersome requirement of lowtemperature. Finally, since p-methoxybenzylthioether acid chloride iscircumvented in the present invention, methylene chloride and the safetyprecautions employed therewith need not be used.

The present invention also improves upon the yield of the reactionbetween the calicheamicin and the linker intermediate compared to theprior processes, such as the process described in U.S. Pat. No.8,273,862. The present invention improves the yield of the resultingcalicheamicin derivative by including a carbodiimide in the reaction.

It has also been discovered, as described herein, that a new method forpurifying calicheamicin derivatives can be achieved, which methodinvolves the use of reversed phase high performance liquidchromatography (RP-HPLC), despite the presence of two water-labilegroups (hydrazone and N-hydroxysuccinimide ester) on the calicheamicinderivative. This invention thereby overcomes the problems mentionedabove associated with purifying calicheamicin derivatives using normalphase chromatography, e.g. using methylene chloride, as described inU.S. Pat. No. 8,273,862.

The present invention, provides compounds of Formula I

wherein R¹² is selected from straight and branched-chain C₁-C₈ alkyl;

each R¹⁰ is independently selected from hydrogen, R¹² and —OR¹²;

R⁸ and R⁹ are each independently selected from hydrogen and straight andbranched-chain C₁-C₈ alkyl, wherein each said alkyl for R⁸ and R⁹ isindependently optionally substituted by —NH₂, —NHR¹¹, —NR¹¹R¹³, —OR¹¹,—OH, or —SR¹¹, wherein each R¹¹ and each R¹³ are independently selectedfrom straight and branched-chain C₁-C₅ alkyl;

r is an integer selected from 0 and 1;

G is oxygen or sulfur;

Z¹ is H or straight or branched-chain C₁-C₅ alkyl;

Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, twoor three groups independently selected from straight or branched-chainC₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴, —C(═O)NHR¹⁴,—O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴, —O(CH₂)_(n)C(═O)NHR¹⁴, and—S(CH₂)_(n)C(═O)NHR¹⁴, or Ar is a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-,1,8-, 2,3-, 2,6-, or 2,7-naphthylidene optionally substituted with one,two, three, or four groups independently selected from straight orbranched-chain C₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴,—C(═O)NHR¹⁴, —O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴,—O(CH₂)_(n)C(═O)NHR¹⁴, and —S(CH₂)_(n)C(═O)NHR¹⁴;

wherein each R¹⁴ is independently selected from (C₁-C₅)alkyl and eachR¹⁴ is independently optionally substituted with one or two groupsselected from —OH, —(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl;

each n is an integer independently selected from 0, 1, 2, 3, 4, and 5;

W is selected from —O—, —S—, —C(═O)NH—, —NHC(═O)—, and —NR¹⁵—, whereinR¹⁵ is a (C₁-C₅)alkyl and R¹⁵ is optionally substituted with one or twogroups selected from —OH, —(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl; and

Y is a straight or branched-chain (C₁-C₆)alkylene group or a straight orbranched-chain (C₂-C₆)alkenylene group.

Compounds of Formula 1 are useful as intermediates for synthesizinglinker intermediates and calicheamicin derivatives comprising suchlinker intermediates, which calicheamicin derivatives can in turn beconjugated to biomacromolecules such as monoclonal antibodies.

In one embodiment of the invention, each R¹⁰ in the compound of FormulaI is hydrogen. In another embodiment, each R¹⁰ in the compound ofFormula I is hydrogen and R¹² is methyl.

In another embodiment of the invention, the compound of Formula I is acompound having the structure

In another embodiment of the invention, R⁸ and R⁹ in the compound ofFormula I are both methyl, r is 0, G is oxygen, Z¹ is methyl, Ar is1,4-phenylene, W is —O—, and Y is —(CH₂)₃—.

In another embodiment of the invention R⁸ and R⁹ in the compound ofFormula I are both methyl.

In another embodiment of the invention, r in the compound of Formula Iis 0. In another embodiment of the invention, r in the compound ofFormula I is 0 and G is oxygen. In another embodiment of the invention,r in the compound of Formula I is 0 and G is sulfur.

The present invention also provides methods for synthesizing theaforementioned compounds of Formula I, which are as stated useful asintermediates for synthesizing linker intermediates and calicheamicinderivatives comprising said linker intermediate groups. In oneembodiment, the present invention provides a method for synthesizing acompound of Formula I

wherein R¹² is selected from straight and branched-chain C₁-C₈ alkyl;

each R¹⁰ is independently selected from hydrogen, R¹² and —OR¹²;

R⁸ and R⁹ are each independently selected from hydrogen and straight andbranched-chain C₁-C₈ alkyl, wherein each said alkyl for R⁸ and R⁹ isindependently optionally substituted by —NH₂, —NHR¹¹, —NR¹¹R¹³, —OR¹¹,—OH, or —SR¹¹, wherein each R¹¹ and each R¹³ are independently selectedfrom straight and branched-chain C₁-C₅ alkyl;

r is an integer selected from 0 and 1;

G is oxygen or sulfur;

Z¹ is H or straight or branched-chain C₁-C₅ alkyl;

Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, twoor three groups independently selected from straight or branched-chainC₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴, —C(═O)NHR¹⁴,—O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴, —O(CH₂)_(n)C(═O)NHR¹⁴, and—S(CH₂)_(n)C(═O)NHR¹⁴, or Ar is a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-,1,8-, 2,3-, 2,6-, or 2,7-naphthylidene optionally substituted with one,two, three, or four groups independently selected from straight orbranched-chain C₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴,—C(═O)NHR¹⁴, —O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴,—O(CH₂)_(n)C(═O)NHR¹⁴, and —S(CH₂)_(n)C(═O)NHR¹⁴;

wherein each R¹⁴ is independently selected from (C₁-C₅)alkyl and eachR¹⁴ is independently optionally substituted with one or two groupsselected from —OH, —(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl;

each n is an integer independently selected from 0, 1, 2, 3, 4, and 5;

W is selected from —O—, —S—, —C(═O)NH—, —NHC(═O)—, and —NR¹⁵—, whereinR¹⁵ is a (C₁-C₅)alkyl and R¹⁵ is optionally substituted with one or twogroups selected from —OH, —(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl; and Y is astraight or branched-chain (C₁-C₆)alkylene group or a straight orbranched-chain (C₂-C₆)alkenylene group; which method comprises reactinga compound of Formula II

wherein R¹⁰, R¹², R⁸ R⁹, r and G are as defined above, with a compoundof Formula III

wherein Z¹, Ar, W and Y are as defined above. In one embodiment of themethod of synthesizing a compound of Formula I, r is 0, G is oxygen, Z¹is methyl, Ar is 1,4-phenylene, W is —O—, and Y is —(CH₂)₃—. In anotherembodiment of the method of synthesizing a compound of Formula I, R⁸ andR⁹ are methyl. In another embodiment of the method of synthesizing acompound of Formula I, each R¹⁰ is hydrogen. In another embodiment ofthe method of synthesizing a compound of Formula I, each R¹⁰ is hydrogenand R¹² is methyl.

In another embodiment of the invention for synthesizing a compound ofFormula I, R⁸ and R⁹ in the compound of Formula II are both methyl.

In another embodiment of the invention for synthesizing a compound ofFormula I, r in the compound of Formula II is 0. In another embodimentof the invention for synthesizing a compound of Formula I, r in thecompound of Formula II is 0 and G is oxygen. In another embodiment ofthe invention for synthesizing a compound of Formula I, r in thecompound of Formula II is 0 and G is sulfur.

The present invention also provides a method of synthesizing a compoundof Formula II

-   -   wherein R¹² is selected from straight and branched-chain C₁-C₈        alkyl;

each R¹⁰ is independently selected from hydrogen, R¹² and —OR¹²;

R⁸ and R⁹ are each independently selected from hydrogen and straight andbranched-chain C₁-C₈ alkyl, wherein each said alkyl for R⁸ and R⁹ isindependently optionally substituted by —NH₂, —NHR¹¹, —NR¹¹R¹³, —OR¹¹,—OH, or —SR¹¹, wherein each R¹¹ and each R¹³ are independently selectedfrom straight and branched-chain C₁-C₅ alkyl;

r is an integer selected from 0 and 1; and

G is oxygen or sulfur;

which method comprises treating a compound of Formula VII

wherein R¹⁶ is —C(═O)OH or —C(═V)SH, wherein V is oxygen or sulfur, orR¹⁶ is —NH₂;

with an azole activating agent of Formula IX

wherein V′ is oxygen or sulfur; and

wherein E is

wherein m is an integer 0, 1, 2, or 3; q is an integer 0, 1 or 2; and pis an integer 0, 1, 2, 3, or 4; and wherein each R¹⁷ attached to E isindependently selected from straight and branched-chain (C₁-C₆)alkylgroups;

in an organic solvent to form a compound of Formula VIII

wherein when R¹⁶ is —C(═O)OH, r is 0 and G is oxygen; when R¹⁶ is—C(═V)SH, r is 0 and G is V; and when R¹⁶ is —NH₂, r is 1 and G is V′;

followed by combining the compound of Formula VIII with hydrazine,thereby forming a compound of Formula II. Compounds of Formula II areuseful as intermediates for synthesizing linker intermediates andcalicheamicin derivatives comprising said linker intermediate groups,which calicheamicin derivatives can in turn be conjugated to abiomacromolecule, such as a monoclonal antibody.

The azole activating agent is any azole-containing compound of the givenFormula IX that will when reacted with a compound of Formula VII afforda compound of Formula VIII wherein E is as described above. Examples ofazole activating agents that can be used in the subject inventioninclude carbonyl diimidazole; thiocarbonyl diimidazole; carbonylbis-pyrazole wherein each pyrazole is optionally substituted with fromone to three (C₁-C₆) alkyl groups; carbonyl bis-1,2,3-triazole; carbonylbis-benzotriazole, and carbonyl bis-1,2,4-triazole. Preferably, theazole activating agent is carbonyl diimidazole.

The compound of Formula VIII is optionally isolated before combiningwith hydrazine. In one embodiment, the compound of Formula VIII is notisolated before combining with hydrazine. In another embodiment, thecompound of Formula VIII is isolated before combining with hydrazine.

Preferably, R¹⁶ is —C(═O)OH and the azole activating agent is carbonyldiimidazole.

In one embodiment of the present method for making a compound of FormulaII, r is 0 and G is oxygen in the compound of Formula II. In anotherembodiment of the present method for making a compound of Formula II, ris 0 and G is oxygen in the compound of Formula II and R¹⁶ in thecompound of Formula VII is —C(═O)OH. In a further embodiment of themethod for making a compound of Formula II, r is 0 and G is oxygen inthe compound of Formula II, R¹⁶ in Formula VII is —C(═O)OH, and theazole activating agent is carbonyl diimidazole.

In another embodiment of the present method for making a compound ofFormula II, the compound of Formula VIII has the structure:

In a further embodiment of the method for making the compound of FormulaII, the compound of Formula VIII has the structure:

and the azole activating agent is carbonyl diimidazole.

In another embodiment of the invention for synthesizing a compound ofFormula II, R⁸ and R⁹ in the compound of Formula VII are both methyl.

In another embodiment of the invention for synthesizing a compound ofFormula II, the compound of Formula VII comprises R¹⁶ being —C(═V)SH andV being oxygen or sulfur. It is to be understood that when the compoundof Formula VII comprises R¹⁶ being —C(═V)SH and V being oxygen, suchcompound of Formula VII can exist in a tautomeric form that is the samecompound of Formula VII but wherein R¹⁶ is —(C═S)OH. When the compoundof Formula VII comprises R¹⁶ being —C(═O)SH, and its tautomer whereinR¹⁶ is —C(═S)OH, the resulting product compound of Formula II comprisesG being oxygen.

In another embodiment of the invention for synthesizing a compound ofFormula II, the compound of Formula VII comprises R¹⁶ being —NH₂. Whenthe compound of Formula VII in the method of synthesizing the compoundof Formula II comprises R¹⁶ being —NH₂, r in the compound of Formula IIresulting from the method is 1. In another embodiment of the method forsynthesizing a compound of Formula II, the method comprises a compoundof Formula VII wherein R¹⁶ is —NH₂, and a compound of Formula IX,wherein V′ is oxygen, the compound of Formula II resulting from saidmethod comprises r being 1 and G being V′ (i.e. oxygen). In anotherembodiment of the method for synthesizing a compound of Formula II, themethod comprises a compound of Formula VII wherein R¹⁶ is —NH₂, and acompound of Formula IX, wherein V′ is sulfur, the compound of Formula IIresulting from said method comprises r being 1 and G being V′ (i.e.sulfur).

In another embodiment of the method for synthesizing a compound ofFormula II, the method comprises an azole activating agent of Formula IXwherein V′ is oxygen. In another embodiment of the method forsynthesizing a compound of Formula II, the method comprises an azoleactivating agent of Formula IX wherein V′ is sulfur.

In another embodiment of the method of synthesizing a compound ofFormula II, the hydrazine is anhydrous hydrazine. In another embodimentof the method of synthesizing a compound of Formula II, the hydrazine ishydrazine monohydrate. In another embodiment, the hydrazine is anaqueous solution of hydrazine. In another embodiment, the hydrazine is atetrahydrofuran solution of hydrazine.

As explained above, compounds of Formula I are useful as intermediatesfor synthesizing linker intermediates and calicheamicin derivativescomprising said linker intermediate groups. Accordingly, the subjectinvention provides a method for synthesizing compounds of Formula IV,which are also useful as intermediates for synthesizing linkerintermediates and calicheamicin derivatives comprising said linkerintermediate groups. In one embodiment, the subject invention provides amethod for synthesizing a compound of Formula IV

wherein R⁸ and R⁹ are each independently selected from hydrogen andstraight and branched-chain C₁-C₈ alkyl, wherein each said alkyl for R⁸and R⁹ is independently optionally substituted by —NH₂, —NHR¹¹,—NR¹¹R¹³, —OR¹¹, —OH, or —SR¹¹, wherein each R¹¹ and each R¹³ areindependently selected from straight and branched-chain C₁-C₅ alkyl;

r is an integer selected from 0 and 1;

G is oxygen or sulfur;

Z¹ is H or straight or branched-chain C₁-C₅ alkyl;

Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, twoor three groups independently selected from straight or branched-chainC₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴, —C(═O)NHR¹⁴,—O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴, —O(CH₂)_(n)C(═O)NHR¹⁴, and—S(CH₂)_(n)C(═O)NHR¹⁴, or Ar is a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-,1,8-, 2,3-, 2,6-, or 2,7-naphthylidene optionally substituted with one,two, three, or four groups independently selected from straight orbranched-chain C₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴,—C(═O)NHR¹⁴, —O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴,—O(CH₂)_(n)C(═O)NHR¹⁴, and —S(CH₂)_(n)C(═O)NHR¹⁴;

wherein each R¹⁴ is independently selected from (C₁-C₅)alkyl and eachR¹⁴ is independently optionally substituted with one or two groupsselected from —OH, —(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl;

each n is an integer independently selected from 0, 1, 2, 3, 4, and 5;

W is selected from —O—, —S—, —C(═O)NH—, —NHC(═O)—, and —NR¹⁵—, whereinR¹⁵ is a (C₁-C₅)alkyl and R¹⁵ is optionally substituted with one or twogroups selected from —OH, —(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl; and Y is astraight or branched-chain (C₁-C₆)alkylene group or a straight orbranched-chain (C₂-C₆)alkenylene group, which method comprises treatinga compound of Formula I

wherein R¹² is selected from straight and branched-chain C₁-C₈ alkyl;

each R¹⁰ is independently selected from hydrogen, R¹² and —OR¹²;

and R⁸, R⁹, r, G, Z¹, Ar, W, and Y are as defined;

with a strong acid to form a mixture comprising the compound of FormulaIV. The strong acid used in the method for synthesizing a compound ofFormula IV can be determined by a person of ordinary skill in the art,as it is any acid that will remove the substituted phenyl-methylenegroup from the sulfur atom, resulting in the compound of Formula IV. Inone embodiment, the strong acid used in the method of the invention forsynthesizing the compound of Formula IV is selected from trifluoroaceticacid, sulfuric acid, triflic acid, HCl, HBr, HI. In one embodiment ofthe method for synthesizing the compound of Formula IV, R⁸ and R⁹ aremethyl, r is 0, G is oxygen, Z¹ is methyl, Ar is 1,4-phenylene, W is—O—, and Y is —(CH₂)₃—. In another embodiment of the method ofsynthesizing a compound of Formula IV, each R¹⁰ is hydrogen. In anotherembodiment of the method of synthesizing the compound of Formula IV,each R¹⁰ is hydrogen and R¹² is methyl.

In another embodiment of the invention for synthesizing a compound ofFormula IV, R⁸ and R⁹ in the compound of Formula I are both methyl.

In another embodiment of the invention for synthesizing a compound ofFormula IV, r in the compound of Formula I is 0. In another embodimentof the invention for synthesizing a compound of Formula IV, r in thecompound of Formula I is 0 and G is oxygen. In another embodiment of theinvention for synthesizing a compound of Formula IV, r in the compoundof Formula I is 0 and G is sulfur.

The invention also provides a method for synthesizing a linkerintermediate of Formula V using the intermediates of Formula I and theintermediates of Formula IV. The invention in one embodiment provides amethod for synthesizing a linker intermediate of Formula V

wherein R⁸ and R⁹ are each independently selected from hydrogen andstraight and branched-chain C₁-C₈ alkyl, wherein each said alkyl for R⁸and R⁹ is independently optionally substituted by —NH₂, —NHR¹¹,—NR¹¹R¹³, —OR¹¹, —OH, or —SR¹¹, wherein each R¹¹ and each R¹³ areindependently selected from straight and branched-chain C₁-C₅ alkyl;

r is an integer selected from 0 and 1;

G is oxygen or sulfur;

Z¹ is H or straight or branched-chain C₁-C₅ alkyl;

Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, twoor three groups independently selected from straight or branched-chainC₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴, —C(═O)NHR¹⁴,—O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴, —O(CH₂)_(n)C(═O)NHR¹⁴, and—S(CH₂)_(n)C(═O)NHR¹⁴, or Ar is a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-,1,8-, 2,3-, 2,6-, or 2,7-naphthylidene optionally substituted with one,two, three, or four groups independently selected from straight orbranched-chain C₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴,—C(═O)NHR¹⁴, —O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴,—O(CH₂)_(n)C(═O)NHR¹⁴, and —S(CH₂)_(n)C(═O)NHR¹⁴;

wherein each R¹⁴ is independently selected from (C₁-C₅)alkyl and eachR¹⁴ is independently optionally substituted with one or two groupsselected from —OH, —(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl;

each n is an integer independently selected from 0, 1, 2, 3, 4, and 5;

W is selected from —O—, —S—, —C(═O)NH—, —NHC(═O)—, and —NR¹⁵—, whereinR¹⁵ is a (C₁-C₅)alkyl and R¹⁵ is optionally substituted with one or twogroups selected from —OH, —(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl;

Y is a straight or branched-chain (C₁-C₆)alkylene group or a straight orbranched-chain (C₂-C₆)alkenylene group; and

Z is selected from the group consisting of

which method comprises

a) treating a compound of Formula I

wherein R¹² is selected from straight and branched-chain C₁-C₈ alkyl,and each R¹⁰ is independently selected from hydrogen, R¹² and —OR¹²;

with a strong acid to form a mixture comprising a compound of Formula IV

and

b) reacting the compound of Formula IV with a compound ZH;

thereby synthesizing the linker intermediate of Formula V. In oneembodiment of the method of synthesizing the linker intermediates ofFormula V, the strong acid is trifluoroacetic acid or sulfuric acid.

In another embodiment of the invention for synthesizing a linkerintermediate of Formula V, R⁸ and R⁹ in the compound of Formula I areboth methyl.

In another embodiment of the invention for synthesizing a linkerintermediate of Formula V, r in the compound of Formula I is 0. Inanother embodiment of the invention for synthesizing a linkerintermediate of Formula V, r in the compound of Formula I is 0 and G isoxygen. In another embodiment of the invention for synthesizing a linerintermediate of Formula V, r in the compound of Formula I is 0 and G issulfur.

In another embodiment of the method of synthesizing the linkerintermediates of Formula V, ZH is

In another embodiment of the method of synthesizing the linkerintermediate of Formula V, the linker intermediate is a compound havingthe structure

In another embodiment of the method of synthesizing the linkerintermediates of Formula V, the compound of Formula I used in the methodis obtained by reacting a compound of Formula II

with a compound of Formula III

As explained, the linker intermediates are useful for preparingcalicheamicin derivatives comprising said linker intermediate groups.The calicheamicin derivatives can in turn be conjugated tobiomacromolecules such as monoclonal antibodies. Accordingly, thecompounds of Formula I and the methods of synthesis described herein areuseful for making such calicheamicin derivatives. The present inventionthus provides a method of synthesizing a calicheamicin derivative ofFormula VI

wherein J is

R₁ is

or CH₃; R₂ is

or H; R₃ is

or H; R₄ is

or H;

R⁵ is —CH₃, —C₂H₅, or —CH(CH₃)₂;

X is an iodine or bromine atom;

R^(5′) is a hydrogen or the group RCO, wherein R is hydrogen, branchedor unbranched alkyl of 1 to 10 carbon atoms, alkylene of 2 to 10 carbonatoms, aryl of 6 to 11 carbon atoms, a (C₆-C₁₁) aryl-alkyl (C₁-C₅)group, or a heteroaryl or heteroaryl-alkyl (C₁-C₅) group whereinheteroaryl is defined as 2- or 3-furyl, 2- or 3-thienyl, 2- or3-(N-methylpyrrolyl), 2-, 3-, or 4-pyridinyl, 2-, 4-, or5-(N-methylimidazolyl), 2-, 4-, or 5-oxazolyl, 2-, 3-, 5-, or6-pyrimidinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolyl, or 1-, 3-, 4-, 5-,6-, 7-, or 8-isoquinolyl, all aryl and heteroaryl groups optionallysubstituted by one or more hydroxy, amino, carboxy, halo, nitro, (C₁-C₃)alkoxy, or thioalkoxy of 1 to 5 carbon atoms;

R₆ and R₇ are each independently selected from H and

R⁸ and R⁹ are each independently selected from hydrogen and straight andbranched-chain C₁-C₈ alkyl, wherein each said alkyl for R⁸ and R⁹ isindependently optionally substituted by —NH₂, —NHR¹¹, —NR¹¹R¹³, —OR¹¹,—OH, or —SR¹¹, wherein each R¹¹ and each R¹³ are independently selectedfrom straight and branched-chain C₁-C₅ alkyl;

r is an integer 0 or 1;

G is oxygen or sulfur;

Z¹ is H or straight or branched-chain C₁-C₅ alkyl;

Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, twoor three groups independently selected from straight or branched-chainC₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴, —C(═O)NHR¹⁴,—O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴, —O(CH₂)_(n)C(═O)NHR¹⁴, and—S(CH₂)_(n)C(═O)NHR¹⁴, or Ar is a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-,1,8-, 2,3-, 2,6-, or 2,7-naphthylidene optionally substituted with one,two, three, or four groups independently selected from straight orbranched-chain C₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴,—C(═O)NHR¹⁴, —O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴,—O(CH₂)_(n)C(═O)NHR¹⁴, and —S(CH₂)_(n)C(═O)NHR¹⁴;

wherein each R¹⁴ is independently selected from (C₁-C₅)alkyl and eachR¹⁴ is independently optionally substituted with one or two groupsselected from —OH, —(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl;

each n is an integer independently selected from 0, 1, 2, 3, 4, and 5;

W is selected from —O—, —S—, —C(═O)NH—, —NHC(═O)—, and —NR¹⁵—, whereinR¹⁵ is a (C₁-C₅)alkyl and R¹⁵ is optionally substituted with one or twogroups selected from —OH, —(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl;

Y is a straight or branched-chain (C₁-C₆)alkylene group or a straight orbranched-chain (C₂-C₆)alkenylene group; and

Z is selected from the group consisting of

which method comprises

a) treating a compound of Formula I

wherein R¹² is selected from straight and branched-chain C₁-C₈ alkyl,

and each R¹⁰ is independently selected from hydrogen, R¹² and —OR¹²;with a strong acid to form a mixture comprising a compound of Formula IV

b) reacting the compound of Formula IV with a compound ZH;

to form a linker intermediate of Formula V

and

c) reacting the linker intermediate of Formula V resulting from step (b)with a methyltrisulfide compound CH₃—S—S—S-J;

thereby synthesizing a calicheamicin derivative of Formula VI. In oneembodiment of the method of synthesizing the calicheamicin derivative ofFormula VI, the strong acid is sulfuric acid or trifluoroacetic acid. Inanother embodiment of the method of synthesizing the calicheamicinderivative of Formula VI, R⁸ and R⁹ are methyl, r is 0, G is oxygen, Z¹is methyl, Ar is 1,4-phenylene, W is —O—, and Y is —(CH₂)₃—. In anotherembodiment of the method of synthesizing a calicheamicin derivativeFormula VI, each R¹⁰ is hydrogen. In another embodiment of the method ofsynthesizing the calicheamicin derivative of Formula VI, each R¹⁰ ishydrogen and R¹² is methyl.

In another embodiment of the invention for synthesizing a calicheamicinderivative of Formula VI, R⁸ and R⁹ in the compound of Formula I areboth methyl.

In another embodiment of the invention for synthesizing a calicheamicinderivative of Formula VI, r in the compound of Formula I is 0. Inanother embodiment of the invention for synthesizing a calicheamicinderivative of Formula VI, r in the compound of Formula I is 0 and G isoxygen. In another embodiment of the invention for synthesizing acalicheamicin derivative of Formula VI, r in the compound of Formula Iis 0 and G is sulfur.

In another embodiment of the method of synthesizing the calicheamicinderivative of Formula VI, the compound of Formula I is obtained byreacting a compound of Formula II

with a compound of Formula III

In another embodiment of the method of synthesizing the calicheamicinderivative of Formula VI, one of R₆ and R₇ is hydrogen and the other ofR₆ and R₇ is

In one embodiment of the intermediates of Formula I and the methods ofsynthesis of the present invention, Z¹ is methyl. In another embodimentof the intermediates and methods of synthesis of the present invention,r is 0 and G is oxygen. In another embodiment of the intermediates andmethods of synthesis of the present invention, Z¹ is methyl, r is 0 andG is oxygen.

In another embodiment of the methods of synthesis of the presentinvention, J is an ozogamicin moiety.

In another embodiment of the methods of synthesis of the presentinvention, the calicheamicin derivative of Formula VI has the structure

The calicheamicin derivatives of Formula VI synthesized from the methodsof the present invention can be conjugated to a biomacromolecule, forexample to a monoclonal antibody. The calicheamicin derivatives ofFormula VI synthesized from the methods of the present invention can forexample be conjugated to the monoclonal anti-CD22 antibody inotuzumab(an antibody specifically binding to the CD22 antigen expressed on thesurface of certain cancer cells) or to the anti-CD33 antibody gemtuzumab(an antibody specifically targeting the anti-CD33 antigen expressed onthe surface of certain cancer cells). The calicheamicin derivatives ofFormula VI synthesized from the methods of the present invention, whenconjugated to a monoclonal antibody, in one embodiment have thestructure:

wherein Ab is a monoclonal antibody. Examples of the monoclonal antibodyAb include, but are not limited to, gemtuzumab and inotuzumab.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used.

The term “calicheamicin derivative”, as used herein, unless otherwiseindicated, refers to a derivative of a compound of the formulaCH₃—S—S—S-J, wherein J is as defined herein, which derivative comprisesa calicheamicin moiety —S—S-J bonded to a linker intermediate group:

wherein R⁸, R⁹, r, G, Z¹, Ar, W, Y and Z are as defined herein. Thecalicheamicin derivative can be further conjugated (i.e. covalentlybonded), at the end containing the —C(═O)Z moiety, to abiomacromolecule, such as a monoclonal antibody. Examples of compoundsof formula CH3-S—S—S-J are described for example in U.S. Pat. No.4,970,198 and U.S. Pat. No. 5,053,394, both of which are incorporatedherein in their entireties by reference. An example of a compoundCH₃—S—S—S-J is the calicheamicin ozogamicin.

The term “linker intermediate”, as used herein, unless otherwiseindicated, refers to those isolated molecules of Formula V describedherein, which are capable of being covalently bonded at one end thereofto a molecule of formula CH₃—S—S—S-J, and which have a functional groupon the other end (the —C(═O)Z end) to which can be covalently bonded abiomacromolecule, such as a monoclonal antibody. The isolated linkerintermediates are useful as components for making calichemicinderivatives and calicheamicin linked to a biomacromolecule such as amonoclonal antibody.

Unless otherwise indicated, the term “alkyl” by itself or as part ofanother term refers to a straight chain or branched, saturated orunsaturated hydrocarbon having the indicated number of carbon atoms(e.g., “C₁-C₈” alkyl refers to an alkyl group having from 1 to 8 carbonatoms). When the number of carbon atoms is not indicated, the alkylgroup has from 1 to 8 carbon atoms (unless it is unsaturated, in whichcase the alkyl group will have from 2 to 8 carbon atoms). Representativestraight chain C₁-C₈ alkyls include, but are not limited to, methyl,ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl and n-octyl; whilebranched C₁-C₈ alkyls include, but are not limited to, -isopropyl,-sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and -2-methylbutyl;unsaturated C₂-C₈ alkyls include, but are not limited to, vinyl, allyl,1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, 1-hexyl,2-hexyl, 3-hexyl, acetylenyl, propynyl, 1-butynyl, 2-butynyl,1-pentynyl, 2-pentynyl and 3-methyl-1-butynyl.

Unless otherwise indicated, “alkylene,” by itself or as part of anotherterm, refers to a saturated branched or straight chain or cyclichydrocarbon radical of the stated number of carbon atoms, having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkane. In oneembodiment, “alkylene” refers to a saturated branched or straight chainalkylene radical of the stated number of carbon atoms having twomonovalent radical centers. Typical alkylene radicals include, but arenot limited to: methylene (—CH₂—), 1,2-ethylene —CH₂CH₂—), 1,3-propylene(—CH₂CH₂CH₂—), 1,4-butylene (—CH₂CH₂CH₂CH₂—), and the like. The term“alkenylene” refers to a branched or straight chain or cyclichydrocarbon radical of the stated number of carbon atoms, but havingleast two carbon atoms connected by a double bond, wherein the radicalhas two monovalent radical centers derived by the removal of twohydrogen atoms from two different carbon atoms of a parent alkane. Inone embodiment, “alkylene” refers to a branched or straight chainradical of the stated number of carbon atoms, but having at least twocarbon atoms connected by a double bond, wherein the radical has twomonovalent radical centers. Examples of alkenylene radicals include, butare not limited to, —CH═CH—, —CH₂CH═CH—, and —CH(CH₃)CH═CH.

Unless otherwise indicated, “aryl,” by itself or as part of anotherterm, means a carbocyclic aromatic hydrocarbon radical of 6-20,preferably 6-14, carbon atoms derived by the removal of one hydrogenatom from each of one or more carbon atoms of a parent aromatic ringsystem. Typical aryl groups include, but are not limited to, radicalsderived from benzene, naphthalene, anthracene, biphenyl, and the like.If indicated, an aryl group herein can be optionally substituted. Theterm “arylene” refers to a divalent radical derived from an aryl groupas defined herein.

“Halogen” refers to a fluorine, chlorine, bromine or iodine atom.

The present invention also provides a method of synthesizing acalicheamicin derivative of Formula VI

wherein J is

R₁ is

or CH₃; R₂ is

or H; R₃ is

or H; R₄ is

or H;

R⁵ is —CH₃, —C₂H₅, or —CH(CH₃)₂;

X is an iodine or bromine atom;

R^(5′) is a hydrogen or the group RCO, wherein R is hydrogen, branchedor unbranched alkyl of 1 to 10 carbon atoms, alkylene of 2 to 10 carbonatoms, aryl of 6 to 11 carbon atoms, a (C₆-C₁₁) aryl-alkyl (C₁-C₅)group, or a heteroaryl or heteroaryl-alkyl (C₁-C₅) group whereinheteroaryl is defined as 2- or 3-furyl, 2- or 3-thienyl, 2- or3-(N-methylpyrrolyl), 2-, 3-, or 4-pyridinyl, 2-, 4-, or5-(N-methylimidazolyl), 2-, 4-, or 5-oxazolyl, 2-, 3-, 5-, or6-pyrimidinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolyl, or 1-, 3-, 4-, 5-,6-, 7-, or 8-isoquinolyl, all aryl and heteroaryl groups optionallysubstituted by one or more hydroxy, amino, carboxy, halo, nitro, (C₁-C₃)alkoxy, or thioalkoxy of 1 to 5 carbon atoms;

R₆ and R₇ are each independently selected from H and

R⁸ and R⁹ are each independently selected from hydrogen and straight andbranched-chain C₁-C₈ alkyl, wherein each said alkyl for R⁸ and R⁹ isindependently optionally substituted by —NH₂, —NHR¹¹, —NR¹¹R¹³, —OR¹¹,—OH, or —SR¹¹, wherein each R¹¹ and each R¹³ are independently selectedfrom straight and branched-chain C₁-C₅ alkyl;

r is an integer 0 or 1;

G is oxygen or sulfur;

Z¹ is H or straight or branched-chain C₁-C₅ alkyl;

Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, twoor three groups independently selected from straight or branched-chainC₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴, —C(═O)NHR¹⁴,—O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴, —O(CH₂)_(n)C(═O)NHR¹⁴, and—S(CH₂)_(n)C(═O)NHR¹⁴, or Ar is a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-,1,8-, 2,3-, 2,6-, or 2,7-naphthylidene optionally substituted with one,two, three, or four groups independently selected from straight orbranched-chain C₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴,—C(═O)NHR¹⁴, —O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴,—O(CH₂)_(n)C(═O)NHR¹⁴, and —S(CH₂)_(n)C(═O)NHR¹⁴;

wherein each R¹⁴ is independently selected from (C₁-C₅)alkyl and eachR¹⁴ is independently optionally substituted with one or two groupsselected from —OH, —(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl;

each n is an integer independently selected from 0, 1, 2, 3, 4, and 5;

W is selected from —O—, —S—, —C(═O)NH—, —NHC(═O)—, and —NR¹⁵—, whereinR¹⁵ is a (C₁-C₅)alkyl and R¹⁵ is optionally substituted with one or twogroups selected from —OH, —(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl;

Y is a straight or branched-chain (C₁-C₆)alkylene group or a straight orbranched-chain (C₂-C₆)alkenylene group; and

Z is selected from the group consisting of

which method comprises reacting a linker intermediate of Formula V

with a methyltrisulfide compound CH₃—S—S—S-J, in the presence of acarbodiimide; thereby synthesizing a calicheamicin derivative of FormulaVI.

The present invention also provides a method of synthesizing acalicheamicin derivative of Formula X

wherein J is

R₁ is

or CH₃; R₂ is

or H; R₃ is

or H; R₄ is

or H;

R⁵ is —CH₃, —O₂H₅, or —CH(CH₃)₂;

X is an iodine or bromine atom;

R^(5′) is a hydrogen or the group RCO, wherein R is hydrogen, branchedor unbranched alkyl of 1 to 10 carbon atoms, alkylene of 2 to 10 carbonatoms, aryl of 6 to 11 carbon atoms, a (C₆-C₁₁) aryl-alkyl (C₁-C₅)group, or a heteroaryl or heteroaryl-alkyl (C₁-C₅) group whereinheteroaryl is defined as 2- or 3-furyl, 2- or 3-thienyl, 2- or3-(N-methylpyrrolyl), 2-, 3-, or 4-pyridinyl, 2-, 4-, or5-(N-methylimidazolyl), 2-, 4-, or 5-oxazolyl, 2-, 3-, 5-, or6-pyrimidinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolyl, or 1-, 3-, 4-, 5-,6-, 7-, or 8-isoquinolyl, all aryl and heteroaryl groups optionallysubstituted by one or more hydroxy, amino, carboxy, halo, nitro, (C₁-C₃)alkoxy, or thioalkoxy of 1 to 5 carbon atoms;

R₆ and R₇ are each independently selected from H and

Y′ is a straight or branched-chain (C₁-C₁₈)alkylene group, a straight orbranched-chain (C₂-C₁₈)alkenylene group, an arylene group, or aheteroarylene group, an arylene (C₁-C₁₈)alkylene group, an arylene(C₂-C₁₈)alkenylene group, a heteroarylene (C₁-C₁₈) alkylene group, or aheteroarylene (C₁-C₁₈)alkenylene group, wherein said heteroarylene groupis a divalent radical derived from furyl, thienyl, N-methylpyrrolyl,pyridinyl, N-methylimidazolyl, oxazolyl, pyrimidinyl, quinolyl,isoquinolyl, N-methylcarbazoyl, aminocoumarinyl, or phenazinyl, andwherein said Y′ can optionally be substituted by dialkylamino of from 1to 5 carbon atoms, alkoxy of from 1 to 5 carbon atoms, hydroxy, —SH, oralkylthio of from 1 to 5 carbon atoms;

Q is selected from —C(═O)NHN═, —C(═S)NHN═, —NHC(═O)NHN═, —NHC(═S)NHN═,and —O—N═;

Z¹ is H or straight or branched-chain C₁-C₅ alkyl;

Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, twoor three groups independently selected from straight or branched-chainC₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴, —C(═O)NHR¹⁴,—O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴, —O(CH₂)_(n)C(═O)NHR¹⁴, and—S(CH₂)_(n)C(═O)NHR¹⁴, or Ar is a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-,1,8-, 2,3-, 2,6-, or 2,7-naphthylidene optionally substituted with one,two, three, or four groups independently selected from straight orbranched-chain C₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴,—C(═O)NHR¹⁴, —O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴,—O(CH₂)_(n)C(═O)NHR¹⁴, and —S(CH₂)_(n)C(═O)NHR¹⁴;

wherein each R¹⁴ is independently selected from (C₁-C₅)alkyl and eachR¹⁴ is independently optionally substituted with one or two groupsselected from —OH, —(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl;

each n is an integer independently selected from 0, 1, 2, 3, 4, and 5;

W is selected from —O—, —S—, —C(═O)NH—, —NHC(═O)—, and —NR¹⁵—, whereinR¹⁵ is a (C₁-C₅)alkyl and R¹⁵ is optionally substituted with one or twogroups selected from —OH, —(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl;

Y is a straight or branched-chain (C₁-C₆)alkylene group or a straight orbranched-chain (C₂-C₆)alkenylene group; and

Z is selected from the group consisting of

which method comprises reacting a linker intermediate of Formula XI

with a methyltrisulfide compound CH₃—S—S—S-J, in the presence of acarbodiimide; thereby synthesizing a calicheamicin derivative of FormulaX.

In one embodiment of this method of synthesizing a calicheamicinderivative of Formula VI or of Formula X in the presence of acarbodiimide, one of R₆ and R₇ is hydrogen and the other of R₆ and R₇ is

In another embodiment of the method of the present invention forsynthesizing a calicheamicin derivative of Formula VI or of Formula X,the methyltrisulfide has an initial concentration in said reaction withthe linker intermediate of Formula V (or of Formula XI as the case maybe) of greater than about 3 g/L of the reaction mixture. In anotherembodiment, the methyltrisulfide compound has an initial concentrationin said reaction with the linker intermediate of Formula V (or ofFormula XI as the case may be) of between about 10 g/L and 110 g/L ofthe reaction mixture.

In another embodiment of this method of synthesizing a calicheamicinderivative of Formula VI or of Formula X, the carbodiimide is1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The carbodiimide in thismethod of the invention for synthesizing a calicheamicin derivative ofFormula VI or Formula X can be any molecule containing a carbodiimidemoiety, and such molecules are known in the art. Examples ofcarbodiimides that can be used in the present invention include, but arenot limited to, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC);N,N′-dicyclohexyl carbodiimide (DCC); N,N′-diisopropyl carbodiimide(DIC); N-cyclohexyl-N′-(2-morpholinoethyl) carbodiimide;N-cyclohexyl-N′-[2-(4-methylmorpholin-4-ium-4-yl)ethyl] carbodiimidetosylate; N-cylcohexyl-N′-[4-(diethylmethylammonio)cyclohexyl]carbodiimide tosylate;N,N′-bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl]carbodiimide; andN-benzyl-N′-isopropylcarbodiimide. Other carbodiimides suitable foremploying in the methods of the present invention can be ascertained bythose of ordinary skill in the art.

The present invention also provides a method for purifying acalicheamicin derivative of Formula VI or a calicheamicin derivative ofFormula X, wherein Formula VI and Formula X are as defined hereinabove,comprising subjecting a calicheamicin derivative of Formula VI or acalicheamicin derivative of Formula X to a reversed phase highperformance liquid chromatography (RP-HPLC) purification protocol. It issurprising that a reversed phase purification protocol can be used forpurifying the calicheamicin derivative molecules of Formula VI orFormula X considering that these compounds have two water-labile groups,namely a hydrazone group and a N-hydroxy succinimide (NHS) ester group,each group having a different pH dependency for its hydrolysis. Yet,using reversed phase purification is advantageous relative to the normalphase chromatography purification described in the prior art (see U.S.Pat. No. 8,273,862), because the normal phase chromatography uses toxic,environmentally-unfriendly solvent, such as methylene chloride andmethanol. The reversed phase high performance liquid chromatography(RP-HPLC) protocol entails use of a C-18 stationary phase that binds thecomponents of the reaction mixture. These components are then eluted andseparated using a gradient consisting of aqueous and organic mobilephases ranging in pH from about 4 to about 6 for optimal stability ofthe two hydrolysable groups present. In one embodiment, the gradientcomprises at least two phases. In another embodiment, the gradientcomprises 1, 2, 3, 4 or 5 phases. In another embodiment, the gradientcomprises 2 or 3 phases. In another embodiment, the gradient comprises 2phases. Each phase can be organic, aqueous, or a combination thereof.The gradient moves through time from aqueous quality towards increasingorganic quality. Aqueous phases known in the art can be used in thesubject invention. Examples of aqueous phases that can be used in thesubject invention include but are not limited to sodium acetate (NaOAc),sodium succinate, N-methyl morpholine, sodium citrate, and2-(N-morpholino)ethanesulfonic acid. Organic phases known in the art canbe used in the subject invention. Examples of organic phases that can beused in the subject invention include but are not limited toacetonitrile, isopropanol, acetone, dimethoxyethane, andN-methyl-2-pyrrolidone. As stated, any of the phases may comprise amixture of aqueous and organic quality, for example a mixture of NaOAcand acetonitrile. For example, a mobile phase consisting of 55% 20 mMsodium acetate, pH 5 and 45% acetonitrile is an aqueous/organic mobilephase that can be used for the RP-HPLC purification for the invention.As a further example, an example of a gradient useful in the subjectinvention is a gradient comprising a first mobile phase consisting of55% 20 mM sodium acetate, pH 5, and 45% acetonitrile, followed by asecond mobile phase consisting of acetonitrile.

In one embodiment of the method of purifying a calicheamicin derivativeof Formula VI or of Formula X comprising a RP-HPLC protocol, thecalicheamicin derivative of Formula VI or of Formula X comprises Z beingselected from:

In another embodiment of the method of purifying a calicheamicinderivative of Formula VI or of Formula X comprising a RP-HPLC protocol,the calicheamicin derivative of Formula VI or of Formula X comprises Zbeing selected from:

In another embodiment of the method of purifying a calicheamicinderivative of Formula VI or of Formula X comprising a RP-HPLC protocol,the calicheamicin derivative of Formula VI or of Formula X comprises Zbeing:

Subsequent to reversed phase purification, the resulting purifiedcalicheamicin derivative can be isolated as described in the art, forexample by concentration and partitioning, or the purified calicheamicinderivative can by isolated by using a solid phase extraction (SPE)protocol. A solid phase extraction protocol was identified as anefficient replacement for the dichloromethane partitioning for productisolation from RP-HPLC fractions. In a solid phase extraction, theproduct is bound to a reversed phase resin by loading in a weak solvent,washed to remove buffer salts (remaining from RP-HPLC purification), andthen eluted with organic solvents, such as acetonitrile, affording aconcentrated product solution free or substantially free of salts.

Thus, further embodiments of any of the methods of the inventiondescribed herein for synthesizing a calicheamicin derivative of FormulaVI or of Formula X, comprise furthermore purifying the synthesizedcalicheamicin derivative of Formula VI or Formula X by subjection to areversed phase purification protocol. In further embodiments of theinvention, a calicheamicin derivative of Formula VI or Formula Xresulting from a reversed phase purification protocol is subsequentlysubjected to a solid phase extraction protocol.

DETAILED DESCRIPTION OF THE INVENTION

The method of the present invention for synthesizing a linkerintermediate of Formula V is described in the following reaction SchemesI-IV. In the chemical formulae in Schemes I-IV, R⁸, R⁹, R¹⁰, R¹², Q, Ar,W, Y, Z¹, and Z are as defined above herein.

Referring to Scheme I, a (4-alkoxyphenyl)methanethiol 1, wherein R¹² isas described herein, is reacted with methyl senecioate 2 to yield thecarboxylic acid intermediate compound 3. Intermediate 3 is then chargedwith a suitable organic solvent such as tetrahydrofuran and an azoleactivating agent of the Formula IX as described herein, such as carbonyldiimidazole (CDI). Intermediate 3.5 is obtained. This reaction isfollowed by combining 3.5 with a hydrazine. The hydrazine source may beanhydrous hydrazine as is described in WO 2008/147765; however,preferably, the hydrazine source is aqueous hydrazine, such as hydrazinemonohydrate. This reaction yields the intermediate compound 4. Thecompound p-methoxybenzylthioether hydrazide, which is an intermediatecompound 4 wherein R¹² is methyl and each R¹⁰ is hydrogen, is describedin WO 2008/147765, the entire contents of which are incorporated hereinby reference.

In Scheme II, intermediate 4 is reacted with the compound 5 wherein Z¹is as described herein, for example 4-(4-acyl-phenoxy)butanoic acid, inan inert (in other words, non-reactive) solvent, optionally with anacidic catalyst, to provide the intermediate 6. Examples of inertsolvents that may be used in this reaction include, but are not limitedto, alcohols (for example, methanol), ethers and esters such as ethylacetate. A person of ordinary skill in the art can determine a suitableinert solvent for this reaction. Acidic catalysts can also be determinedby a person of ordinary skill in the art; examples of acidic catalystsinclude, but are not limited to acetic acid.

In Scheme III, intermediate compound 6 is deprotected to form compound7. In this reaction, compound 6 is charged with methoxybenzene and astrong acid optionally under heat, for example trifluoroacetic acidunder heat, yielding intermediate compound 7. Other strong acids may beused instead of trifluoroacetic acid, for example sulfuric acid.

In Scheme IV, intermediate 7 is converted to a compound 8, which is anembodiment of the linker intermediate of Formula V described herein.Intermediate 7 can be converted to a linker intermediate such as 8 as isdescribed in the art, such as in U.S. Pat. No. 8,273,862, which isincorporated herein by reference in its entirety. Preferably, however,as depicted in Scheme IV, intermediate 7 is reacted with a tertiaryamine base such as triethylamine (TEA) and with trimethylacetyl chloride(PivCl) in the presence of an inert solvent such as tetrahydrofuran.Subsequently, a compound of Formula ZH, for exampleN-hydroxysuccinimide, is introduced to provide the linker intermediate8.

After synthesis of the linker intermediate of Formula V (such as acompound 8), a calicheamicin derivative of Formula VI is thensynthesized using the linker intermediate from the reaction of Scheme IVand methods known in the art, for example as is described in U.S. Pat.No. 8,273,862. For example, the linker intermediate of Formula V can bereacted first with an alkali methyl carbonate, which includes but is notlimited to sodium carbonate, forming the sodium salt of the linkerintermediate in acetonitrile by heating at gentle reflux. Furtherreaction of the sodium salt of the linker intermediate with themethyltrisulfide CH₃—S—S—S-J at about −15° C., in an inert organicsolvent, preferably acetonitrile gives the calicheamicin derivative ofFormula VI. Preferred is the reaction in acetonitrile at about 0° C.Optionally an organic base may replace the alkali metal carbonate whichpreferably includes triethylamine, in acetonitrile at about 0° C.

Alternatively, a calicheamicin derivative of Formula VI can besynthesized from the linker intermediate of Formula V by reaction with amolecule of formula CH₃—S—S—S-J by using the method described hereincomprising a carbodiimide.

The calicheamicin derivative of Formula VI can be further conjugated toa biomacromolecule, such as a monoclonal antibody, to form anantibody-drug conjugate, using techniques described in the art, forexample the methods described in U.S. Pat. No. 5,053,394, and in U.S.Pat. No. 5,770,701, both of which are incorporated by reference hereinin their entireties.

The following examples are presented to illustrate certain embodimentsof the present invention, but should not be construed as limiting thescope of this invention. Those skilled in the art will readilyunderstand that known variations of the specific conditions of thefollowing examples can be used.

Example 1 3-(4-methoxybenzylthio)-3-methylbutanoic Acid

85 g of 1 and 255 mL of 2-Methyltetrahydrofuran were added to a reactorat 20-25° C. 125.05 g of 2 was added to the reactor and the reactor wasdegassed by bubbling a nitrogen stream into the stirred solution for15-20 min. Tetrabutylammonium fluoride (1M in THF, 0.05 equiv., 36.1 mL)was added to the reactor and the reaction was maintained at 20-30° C.for 2 hours.

A solution of calcium chloride dihydrate (0.35 equiv., 28.060 g) in 255mL of water was added. After stirring for 20 minutes the lower aqueousphase was removed. To the upper organic phase was added 252 mL ofmethanol and 3 equiv. of NaOH in 252 mL of water. The reaction mixturewas stirred until complete consumption of the intermediate ester (3) isobserved.

The reaction was cooled to 15° C. and 2-methyltetrahydrofuran was added(252 mL) followed by 252 mL of water. Concentrated HCl was added (3.1equiv., 184 mL) slowly, maintaining the reaction in the range 15-30° C.The lower aqueous phase was removed. The organic layer was washed with1M HCl (252 mL) and heptanes was added (1940 mL). The solution wasdistilled to remove 2-methyltetrahydrofuran. The resulting slurry wasstirred for an hour, then filtered and dried. The mother liquor wasconcentrated to give a second crop of solids.

Total yield of the title compound acid 4 was 162.59 g (88.5%). ¹H NMR(CDCl₃): δ (ppm) 1.5 (s, 6H), 2.3 (s, 2H), 3.6 (m, 5H), 6.7 (d, 2H), 7.6(d, 2H).

Example 2 3-(4-methoxybenzylthio)-3-methylbutanehydrazide

Acid 4 (81.67 g, 321 mmol) was added to 375 mL of THF (tetrahydrofuran).CDI (1.05 eq., 54.7 g) was charged in three portions and the reactionwas stirred at 20° C. for 2.5 hours. In a separate reactor a solution ofhydrazine monohydrate (2.5 equiv., 40.19 g) in 200 mL of THF wasprepared. The solution of intermediate 5 was added to the hydrazinehydrate solution keeping the internal temperature at 20° C. After theaddition was complete the reaction was stirred for 18 hours, thenconcentrated to 100 mL. 850 mL of EtOAc (ethyl acetate) was added, andthe solution was washed 3 times with 500 mL of water then 200 mL ofbrine. The organic layer was dried with Na₂SO₄, filtered throughdiatomaceous earth and concentrated on a rotary evaporator to a whiteslurry. 300 mL of heptane was added and 200 mL was removed on a rotaryevaporator. Another 200 mL of heptane was added and stripped to a thickwhite slurry. The mixture was filtered, washed with heptanes and dried.83.15 g (96.5%) of the title compound 6 was obtained from 4. MS 269(M+1), 121, 120.

Example 34-(4-(1-(2-(3-(4-methoxybenzylthio)-3-methylbutanoyl)hydrazono)-ethyl)phenoxy)butanoicAcid

68 g of 6 and 57.43 g of 7 were added to 680 mL of methanol (MeOH). 68mL of acetic acid (HOAc) was added and the mixture was heated at 45° C.for 3 hours, cooled to 20° C., and held for 16 hours. The slurry wasfiltered, washed with methanol and dried. 112.60 g of the title compound8 as a mixture of E and Z isomers was obtained. ¹H NMR (DMSO-d₆): δ(ppm) 1.5 (m, 6H), 2.0 (m, 2H), 2.2 (m, 3H), 2.4 (m, 2H), 2.7 (s, 1.1H),3.0 (s, 0.9H), 3.7 (m, 3H), 3.8 (m, 2H), 4.0 (m, 2H), 6.8 (m 2H), 6.9(m, 2H), 7.3 (m, 2H), 7.7 (m, 2H), 10.2&10.3 (s, 1H), 12.1 (s, 1H).LC-MS m/z 473 [M+H]⁺.

Example 44-(4-(1-(2-(3-mercapto-3-methylbutanoyl)hydrazono)ethyl)-phenoxy)butanoicAcid

290.5 g, (614.7 mmol) of 8 and 1162 mL of anisole were charged to areactor at 20-25° C. Trifluoroacetic acid (1162 mL) was added over 2minutes, and the reaction was heated to 65-70° C. for 2.5 hours. Thereaction was cooled to 40° C. The TFA (trifluoroacetic acid) was vacuumdistilled and replaced with 2-methyltetrahydrofuran (2905 mL). Thedistillation was continued until a thick slurry was observed (finalvolume of the slurry was approximately 2 L). The slurry was cooled to15° C. and filtered. The crude product was reslurried in methanol(1836.6 mL), heated to 55° C. and then cooled to 20° C. overnight. Theslurry was filtered, washed with methanol and dried. 171.80 g (93.54%)of the title compound 9 as an E and Z mixture of isomers was obtainedfrom 8. ¹H NMR (DMSO-d₆): δ (ppm) 1.5 (m, 6H), 2.0 (m, 2H), 2.2 (m, 3H),2.4 (m, 2H), 2.7 (s, 1.1H), 3.0 (s, 0.9H), 3.3 (s 1H), 4.0 (m, 2H), 6.9(m, 2H), 7.7 (m, 2H), 10.2&10.3 (s, 1H), 12.1 (s, 1H).

Example 5 2,5-dioxopyrrolidin-1-yl4-(4-(1-(2-(3-mercapto-3-methylbutanoyl)-hydrazono)ethyl)phenoxy)butanoate

Reactor setup: 2-L jacketed reactor, Tr probe, nitrogen inlet.

60 g (170.3 mmol) was added to 2400 mL of THF and cooled to 10° C.Triethylamine (2 equiv., 34.46 g) was added, then trimethylacetylchloride (1.1 equiv., 22.81 g) was added slowly over 10 minutes,maintaining the temperature in the range 10-20° C. The mixture wasstirred at 10-20° C. for 30 minutes. N-hydroxysuccinimide (1.1 equiv.,21.99 g) was added to the reactor and stirred at 20-25° C. for 30minutes.

The slurry was filtered to remove the TEA-HCl salts and concentratedunder vacuum to a volume of approximately 800 mL. Hexane (780 mL) wasslowly added to crystallize the product. The slurry was stirred for 1.5hours, then filtered, washed with heptanes and dried. 70.4 g (92% yield)of the title compound linker intermediate 10 was obtained from 9.

The crude product was recrystallized by adding 144 g of 10 to 2100 mL ofTHF and heated to 60° C. The mixture was filtered through diatomaceouseart, and 2100 mL of hexane slowly added and cooled to 20° C. over 1.5hours. The slurry was filtered, washed with cold THF/hexane (1:1), thenhexane and dried. 126 g (87.5% recovery) of the title compound linkerintermediate 10 was obtained from 9. ¹H NMR (DMSO-d₆): δ (ppm) 1.5 (m,6H), 2.1 (m, 2H), 2.2 (m, 3H), 2.7 (s, 1.1H), 2.9 (M, 5H) 3.0 (s, 1.9H),3.3 (s 1H), 4.3 (m, 2H), 7.0 (m, 2H), 7.5 (m, 2H), 10.2&10.3 (s, 1H).

Example 6 Preparation of Butanoic Acid,3-[[(2E)-2-[(1R,4Z,8S)-8-[[2-O-[4-(acetylethylamino)-2,4-dideoxy-3-O-methyl-a-L-threo-pentopyranosyl]-4,6-dideoxy-4-[[[2,6-dideoxy-4-S-[4-[(6-deoxy-3-O-methyl-a-L-mannopyranosyl)oxy]-3-iodo-5,6-dimethoxy-2-methylbenzoyl]-4-thio-ß-D-ribo-hexopyranosyl]oxy]amino]-ß-D-glucopyranosyl]oxy]-1-hydroxy-10-[(methoxycarbonyl)amino]-11-oxobicyclo[7.3.1]trideca-4,9-diene-2,6-diyn-13-ylidene]ethyl]dithio]-3-methyl-,2-[(1E)-1-[4-[4-[(2,5-dioxo-1-pyrrolidinyl)oxy]-4-oxobutoxyl]phenyl]ethylidene]hydrazide

To a solution of N-acetyl-calicheamicin (50 mg, 0.035 mmol) inacetonitrile (1.0 mL) at room temperature was added linker intermediatebutanoic acid,3-mercapto-3-methyl-,2-[(E)-1-[4-[4-[(2,5-dioxo-1-pyrrolidinyl)-oxy]-4-oxobutoxy]phenyl]ethylidene]hydrazide(31.9 mg, 0.07 mmol) in one portion followed by1,(3-dimethylaminopropyl)-3-ethylcarbodiimide (6.7 mg, 0.035 mmol) andthen triethylamine (5.3 mg, 7.3 μL, 0.053 mmol). The reaction mixture (aslurry) was stirred for 1 hour at room temperature at which point itbecame a yellow solution. The yield was determined by area percentrelative to a derivative standard (60% yield).

Mass Spec:

(M+Na)=1801.4578

¹H NMR:

¹H NMR (CDCl₃+5% CD₃OD, 400 MHz): 8.69 (s, 1H, 18C—NH—N_(═)), 7.75 (d,J=8.7 Hz, 2H, 22), 6.95 (d, J=8.7 Hz, 2H, 23), 6.38 (br t, J=7 Hz, 1H,14), 6.23 (d, J=1.5 Hz, 1H, 8), 5.90 (d, J=9.5 Hz, 1H, 4), 5.80 (dd,J=9.5, 1.5 Hz, 1H, 5), 5.72 (d, J=1.5 Hz, 1H, 1D), 5.63 (br d, J=2.2 Hz,1H, 1E), 5.03 (dd, J=11.5, 1.6 Hz, 1H, 1B), 4.70 (m, 1H, 5E), 4.61 (d,J=7.8 Hz, 1H, 1A), 4.6 (m, 1H, 3E), 4.49 (m, 1H, 2D), 4.31 (m, 1H, 3B),4.20 (m, 1H, 5D), 4.10 (m, 2H, 25), 4.07 (m, 1H, 5B), 4.03 (m, 1H, 3A),3.91 (m, 1H, 15), 3.89 (s, 3H, 2C—OCH₃), 3.84 (s, 3H, 3C—OCH₃), 3.8 (m,1H, 3D), 3.76 (m, 1H, 15), 3.75 (m, 1H, 4B), 3.65 (m, 1H, 5A), 3.63 (bs,3H, 10-NHCOOCH₃), 3.62 (m, 1H, 2A), 3.6 (m, 1H, 4D), 3.57 (s, 3H,3D-OCH₃), 3.4 (m, 1H, 5E), 3.37 (s, 3H, 3E-OCH₃), 3.30 (m, 2H,4E-N—CH₂CH₃), 3.12 (d, J=17.6 Hz, 1H, 12), 3.0 (m, 1H, 4E), 2.85 (m, 2H,27), 2.85 (bs, 4, 30), 2.72 (d, J=17.6 Hz, 1H, 12), 2.5 (m, 2H, 17), 2.4(m, 1H, 2E equatorial), 2.33 (m, 1H, 4A), 2.25 (m, 2H, 26), 2.18 (s, 3H,19), 2.08 (s, 3H, 4E-N—COCH₃), 2.0 (m, 1H, 2B), 1.8 (m, 1H, 2B), 1.50(s, 3H, 16a), 1.44 (s, 3H, 16b), 1.42 (d, J=6.2 Hz, 3H, 6B), 1.4 (m, 1H,2E axial), 1.31 (d, J=6.1 Hz, 3H, 6A), 1.31 (d, J=6.1 Hz, 3H, 6D), 1.19(t, J=7.2 Hz, 3H, 4E-N—CH₂CH₃).

Example 7 Preparation of Butanoic Acid,3-[[(2E)-2-[(1R,4Z,8S)-8-[[2-O-[4-(acetylethylamino)-2,4-dideoxy-3-O-methyl-a-L-threo-pentopyranosyl]-4,6-dideoxy-4-[[[2,6-dideoxy-4-S-[4-[(6-deoxy-3-O-methyl-a-L-mannopyranosyl)oxy]-3-iodo-5,6-dimethoxy-2-methylbenzoyl]-4-thio-ß-D-ribo-hexopyranosyl]oxy]amino]-ß-D-glucopyranosyl]oxy]-1-hydroxy-10-[(methoxycarbonyl)amino]-11-oxobicyclo[7.3.1]trideca-4,9-diene-2,6-diyn-13-ylidene]ethyl]dithio]-3-methyl-,2-[(1E)-1-[4-[4-[(2,5-dioxo-1-pyrrolidinyl)oxy]-4-oxobutoxy]phenyl]ethylidene]hydrazide,Without Using EDC

As a comparison to the method of the present invention illustrated inExample 6, the following reaction was conducted: To a solution ofN-acetyl-calicheamicin (50 mg, 0.035 mmol) in acetonitrile (1.0 mL) atroom temperature was added linker intermediate butanoic acid,3-mercapto-3-methyl-,2-[(E)-1-[4-[4-[(2,5-dioxo-1-pyrrolidinyl)oxy]-4-oxobutoxy]phenyl]-ethylidene]-hydrazide(31.9 mg, 0.07 mmol) in one portion followed by triethylamine (5.3 mg,7.3 uL, 0.053 mmol). The reaction mixture (a slurry) was stirred for 1hour at room temperature at which point it became a yellow solution. Theyield was determined by area percent relative to a derivative standard(35% yield).

Mass Spec:

(M+Na)=1801.4578

¹H NMR:

¹H NMR (CDCl₃+5% CD₃OD, 400 MHz): 8.69 (s, 1H, 18C—NH—N_(═)), 7.75 (d,J=8.7 Hz, 2H, 22), 6.95 (d, J=8.7 Hz, 2H, 23), 6.38 (br t, J=7 Hz, 1H,14), 6.23 (d, J=1.5 Hz, 1H, 8), 5.90 (d, J=9.5 Hz, 1H, 4), 5.80 (dd,J=9.5, 1.5 Hz, 1H, 5), 5.72 (d, J=1.5 Hz, 1H, 1D), 5.63 (br d, J=2.2 Hz,1H, 1E), 5.03 (dd, J=11.5, 1.6 Hz, 1H, 1B), 4.70 (m, 1H, 5E), 4.61 (d,J=7.8 Hz, 1H, 1A), 4.6 (m, 1H, 3E), 4.49 (m, 1H, 2D), 4.31 (m, 1H, 3B),4.20 (m, 1H, 5D), 4.10 (m, 2H, 25), 4.07 (m, 1H, 5B), 4.03 (m, 1H, 3A),3.91 (m, 1H, 15), 3.89 (s, 3H, 2C—OCH₃), 3.84 (s, 3H, 3C—OCH₃), 3.8 (m,1H, 3D), 3.76 (m, 1H, 15), 3.75 (m, 1H, 4B), 3.65 (m, 1H, 5A), 3.63 (bs,3H, 10-NHCOOCH₃), 3.62 (m, 1H, 2A), 3.6 (m, 1H, 4D), 3.57 (s, 3H,3D-OCH₃), 3.4 (m, 1H, 5E), 3.37 (s, 3H, 3E-OCH₃), 3.30 (m, 2H,4E-N—CH₂CH₃), 3.12 (d, J=17.6 Hz, 1H, 12), 3.0 (m, 1H, 4E), 2.85 (m, 2H,27), 2.85 (bs, 4, 30), 2.72 (d, J=17.6 Hz, 1H, 12), 2.5 (m, 2H, 17), 2.4(m, 1H, 2E equatorial), 2.33 (m, 1H, 4A), 2.25 (m, 2H, 26), 2.18 (s, 3H,19), 2.08 (s, 3H, 4E-N—COCH₃), 2.0 (m, 1H, 2B), 1.8 (m, 1H, 2B), 1.50(s, 3H, 16a), 1.44 (s, 3H, 16b), 1.42 (d, J=6.2 Hz, 3H, 6B), 1.4 (m, 1H,2E axial), 1.31 (d, J=6.1 Hz, 3H, 6A), 1.31 (d, J=6.1 Hz, 3H, 6D), 1.19(t, J=7.2 Hz, 3H, 4E-N—CH₂CH₃)

Example 8 Large Scale Preparation of Butanoic Acid,3-[[(2E)-2-[(1R,4Z,8S)-8-[[2-O-[4-(acetylethylamino)-2,4-dideoxy-3-O-methyl-a-L-threo-pentopyranosyl]-4,6-dideoxy-4-[[[2,6-dideoxy-4-S-[4-[(6-deoxy-3-O-methyl-a-L-mannopyranosyl)oxy]-3-iodo-5,6-dimethoxy-2-methylbenzoyl]-4-thio-ß-D-ribo-hexopyranosyl]oxy]amino]-ß-D-glucopyranosyl]oxy]-1-hydroxy-10-[(methoxycarbonyl)amino]-11-oxobicyclo[7.3.1]trideca-4,9-diene-2,6-diyn-13-ylidene]ethyl]dithio]-3-methyl-,2-[(1E)-1-[4-[4-[(2,5-dioxo-1-pyrrolidinyl)oxy]-4-oxobutoxy]phenyl]ethylidene]hydrazide,Followed by Purification

To a solution of N-acetyl-calicheamicin (60.2 g, 42.7 mmol) inacetonitrile (900 mL) at 4° C. was added linker intermediate butanoicacid,3-mercapto-3-methyl-,2-[(E)-1-[4-[4-[(2,5-dioxo-1-pyrrolidinyl)oxy]-4-oxobutoxy]-phenyl]ethylidene]hydrazide(38.4 g, 85.4 mmol) in one portion followed by1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (8.2 g, 42.7 mmol). Thebottles containing linker and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were rinsed withacetonitrile (300 mL) and added to reaction mixture. Triethylamine (6.5g, 8.9 mL, 64.1 mmol) was then added to reaction mixture. The reactionmixture (a slurry) was stirred for 1 hour at 4° C. at which point itbecomes a yellow solution. The reaction mixture was further diluted withacetonitrile (600 mL) and 20 mM sodium acetate buffer, pH 5.0 (1200 mL)and purified by a reversed phase HPLC column using a gradient of mobilephases A and B. (Mobile phase A: 55:45 (v/v) 20 mM NaOAc buffer pH 5.0(pH 4.5-5.5): acetonitrile: Mobile phase B: acetonitrile.) The fractionsof desired purity were pooled and then subjected to solid phaseextraction (SPE). In SPE the purified fractions were loaded onto thecolumn and then washed with a water/acetonitrile mixture and then elutedwith acetonitrile to yield concentrated fractions containing product.The resulting fractions were concentrated in vacuo, taken up in ethylacetate and precipitated by adding hexane. The solids were filtered anddried to provide the title compound as a white solid of 96.9% purity byHPLC. (45.2 g, 60% yield).

Mass Spec:

(M+Na)=1801.4578

¹H NMR:

¹H NMR (CDCl₃+5% CD₃OD, 400 MHz): 8.69 (s, 1H, 18C—NH—N_(═)), 7.75 (d,J=8.7 Hz, 2H, 22), 6.95 (d, J=8.7 Hz, 2H, 23), 6.38 (br t, J=7 Hz, 1H,14), 6.23 (d, J=1.5 Hz, 1H, 8), 5.90 (d, J=9.5 Hz, 1H, 4), 5.80 (dd,J=9.5, 1.5 Hz, 1H, 5), 5.72 (d, J=1.5 Hz, 1H, 1D), 5.63 (br d, J=2.2 Hz,1H, 1E), 5.03 (dd, J=11.5, 1.6 Hz, 1H, 1B), 4.70 (m, 1H, 5E), 4.61 (d,J=7.8 Hz, 1H, 1A), 4.6 (m, 1H, 3E), 4.49 (m, 1H, 2D), 4.31 (m, 1H, 3B),4.20 (m, 1H, 5D), 4.10 (m, 2H, 25), 4.07 (m, 1H, 5B), 4.03 (m, 1H, 3A),3.91 (m, 1H, 15), 3.89 (s, 3H, 2C—OCH₃), 3.84 (s, 3H, 3C—OCH₃), 3.8 (m,1H, 3D), 3.76 (m, 1H, 15), 3.75 (m, 1H, 4B), 3.65 (m, 1H, 5A), 3.63 (bs,3H, 10-NHCOOCH₃), 3.62 (m, 1H, 2A), 3.6 (m, 1H, 4D), 3.57 (s, 3H,3D-OCH₃), 3.4 (m, 1H, 5E), 3.37 (s, 3H, 3E-OCH₃), 3.30 (m, 2H,4E-N—CH₂CH₃), 3.12 (d, J=17.6 Hz, 1H, 12), 3.0 (m, 1H, 4E), 2.85 (m, 2H,27), 2.85 (bs, 4, 30), 2.72 (d, J=17.6 Hz, 1H, 12), 2.5 (m, 2H, 17), 2.4(m, 1H, 2E equatorial), 2.33 (m, 1H, 4A), 2.25 (m, 2H, 26), 2.18 (s, 3H,19), 2.08 (s, 3H, 4E-N—COCH₃), 2.0 (m, 1H, 2B), 1.8 (m, 1H, 2B), 1.50(s, 3H, 16a), 1.44 (s, 3H, 16b), 1.42 (d, J=6.2 Hz, 3H, 6B), 1.4 (m, 1H,2E axial), 1.31 (d, J=6.1 Hz, 3H, 6A), 1.31 (d, J=6.1 Hz, 3H, 6D), 1.19(t, J=7.2 Hz, 3H, 4E-N—CH₂CH₃)

1-91. (canceled)
 92. A method for synthesizing a compound of Formula I

wherein R¹² is selected from straight and branched-chain C₁-C₈ alkyl;each R¹⁰ is independently selected from hydrogen, R¹² and —OR¹²; R⁸ andR⁹ are each independently selected from hydrogen and straight andbranched-chain C₁-C₈ alkyl, wherein each said alkyl for R⁸ and R⁹ isindependently optionally substituted by —NH₂, —NHR¹¹, —NR¹¹R¹³, —OR¹¹,—OH, or —SR¹¹, wherein each R¹¹ and each R¹³ are independently selectedfrom straight and branched-chain C₁-C₅ alkyl; r is an integer 0 or 1; Gis oxygen or sulfur; Z¹ is H or straight or branched-chain C₁-C₅ alkyl;Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, twoor three groups independently selected from straight or branched-chainC₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴, —C(═O)NHR¹⁴,—O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴, —O(CH₂)_(n)C(═O)NHR¹⁴, and—S(CH₂)_(n)C(═O)NHR¹⁴, or Ar is a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-,1,8-, 2,3-, 2,6-, or 2,7-naphthylidene optionally substituted with one,two, three, or four groups independently selected from straight orbranched-chain C₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴,—C(═O)NHR¹⁴, —O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴,—O(CH₂)_(n)C(═O)NHR¹⁴, and —S(CH₂)_(n)C(═O)NHR¹⁴; wherein each R¹⁴ isindependently selected from (C₁-C₅)alkyl and each R¹⁴ is independentlyoptionally substituted with one or two groups selected from —OH,—(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl; each n is an integer independentlyselected from 0, 1, 2, 3, 4, and 5; W is selected from —O—, —S—,—C(═O)NH—, —NHC(═O)—, and —NR¹⁵—, wherein R¹⁵ is a (C₁-C₅)alkyl and R¹⁵is optionally substituted with one or two groups selected from —OH,—(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl; and Y is a straight or branched-chain(C₁-C₆)alkylene group or a straight or branched-chain (C₂-C₆)alkenylenegroup; which method comprises reacting a compound of Formula II

with a compound of Formula III


93. A method according to claim 92, wherein r is 0, G is oxygen, Z¹ ismethyl, Ar is 1,4-phenylene, W is —O—, and Y is —(CH₂)₃—.
 94. A methodaccording to claim 92, wherein R⁸ and R⁹ are methyl.
 95. A methodaccording to claim 92, wherein each R¹⁰ is hydrogen.
 96. A methodaccording to claim 95, wherein R¹² is methyl.
 97. A method forsynthesizing a compound of Formula IV

which method comprises treating a compound of Formula I

wherein R¹² is selected from straight and branched-chain C₁-C₈ alkyl;each R¹⁰ is independently selected from hydrogen, R¹² and —OR¹²; R⁸ andR⁹ are each independently selected from hydrogen and straight andbranched-chain C₁-C₈ alkyl, wherein each said alkyl for R⁸ and R⁹ isindependently optionally substituted by —NH₂, —NHR¹¹, —NR¹¹R¹³, —OR¹¹,—OH, or —SR¹¹, wherein each R¹¹ and each R¹³ are independently selectedfrom straight and branched-chain C₁-C₅ alkyl; r is an integer 0 or 1; Gis oxygen or sulfur; Z¹ is H or straight or branched-chain C₁-C₅ alkyl;Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, twoor three groups independently selected from straight or branched-chainC₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴, —C(═O)NHR¹⁴,—O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴, —O(CH₂)_(n)C(═O)NHR¹⁴, and—S(CH₂)_(n)C(═O)NHR¹⁴, or Ar is a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-,1,8-, 2,3-, 2,6-, or 2,7-naphthylidene optionally substituted with one,two, three, or four groups independently selected from straight orbranched-chain C₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴,—C(═O)NHR¹⁴, —O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴,—O(CH₂)_(n)C(═O)NHR¹⁴, and —S(CH₂)_(n)C(═O)NHR¹⁴; wherein each R¹⁴ isindependently selected from (C₁-C₅)alkyl and each R¹⁴ is independentlyoptionally substituted with one or two groups selected from —OH,—(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl; each n is an integer independentlyselected from 0, 1, 2, 3, 4, and 5; W is selected from —O—, —S—,—C(═O)NH—, —NHC(═O)—, and —NR¹⁵—, wherein R¹⁵ is a (C₁-C₅)alkyl and R¹⁵is optionally substituted with one or two groups selected from —OH,—(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl; and Y is a straight or branched-chain(C₁-C₆)alkylene group or a straight or branched-chain (C₂-C₆)alkenylenegroup, with a strong acid to form a mixture comprising the compound ofFormula IV.
 98. A method according to claim 97, wherein the strong acidis trifluoroacetic acid or sulfuric acid.
 99. A method according toclaim 97, wherein R⁸ and R⁹ are methyl, r is 0, G is oxygen, Z¹ ismethyl, Ar is 1,4-phenylene, W is —O—, and Y is —(CH₂)₃—.
 100. A methodaccording to claim 97, wherein each R¹⁰ is hydrogen.
 101. A methodaccording to claim 100, wherein R¹² is methyl.
 102. A method forsynthesizing a linker intermediate of Formula V

wherein R⁸ and R⁹ are each independently selected from hydrogen andstraight and branched-chain C₁-C₈ alkyl, wherein each said alkyl for R⁸and R⁹ is independently optionally substituted by —NH₂, —NHR¹¹,—NR¹¹R¹³, —OR¹¹, —OH, or —SR¹¹, wherein each R¹¹ and each R¹³ areindependently selected from straight and branched-chain C₁-C₅ alkyl; ris an integer selected from 0 and 1; G is oxygen or sulfur; Z¹ is H orstraight or branched-chain C₁-C₅ alkyl; Ar is 1,2-, 1,3-, or1,4-phenylene optionally substituted with one, two or three groupsindependently selected from straight or branched-chain C₁-C₆ alkyl,—OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴, —C(═O)NHR¹⁴, —O(CH₂)_(n)COOR¹⁴,—S(CH₂)_(n)COOR¹⁴, —O(CH₂)_(n)C(═O)NHR¹⁴, and —S(CH₂)_(n)C(═O)NHR¹⁴, orAr is a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-, 2,6-, or2,7-naphthylidene optionally substituted with one, two, three, or fourgroups independently selected from straight or branched-chain C₁-C₆alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴, —C(═O)NHR¹⁴,—O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴, —O(CH₂)_(n)C(═O)NHR¹⁴, and—S(CH₂)_(n)C(═O)NHR¹⁴; wherein each R¹⁴ is independently selected from(C₁-C₅)alkyl and each R¹⁴ is independently optionally substituted withone or two groups selected from —OH, —(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl;each n is an integer independently selected from 0, 1, 2, 3, 4, and 5; Wis selected from —O—, —S—, —C(═O)NH—, —NHC(═O)—, and —NR¹⁵—, wherein R¹⁵is a (C₁-C₅)alkyl and R¹⁵ is optionally substituted with one or twogroups selected from —OH, —(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl; Y is astraight or branched-chain (C₁-C₆)alkylene group or a straight orbranched-chain (C₂-C₆)alkylene group; and Z is selected from the groupconsisting of

which method comprises a) treating a compound of Formula I

wherein R¹² is selected from straight and branched-chain C₁-C₈ alkyl,and each R¹⁰ is independently selected from hydrogen, R¹² and —OR¹²;with a strong acid to form a mixture comprising a compound of Formula IV

b) reacting the compound of Formula IV with a compound ZH; therebysynthesizing the linker intermediate of Formula V.
 103. A methodaccording to claim 102, wherein the strong acid is trifluoroacetic acidor sulfuric acid.
 104. A method according to claim 102, wherein ZH is


105. A method according to claim 102, wherein the linker intermediate isof the structure


106. A method according to claim 102, wherein the compound of Formula Iis obtained by reacting a compound of Formula II

with a compound of Formula III


107. A method of synthesizing a calicheamicin derivative of Formula VI

wherein J is

R₁ is

or CH₃; R₂ is

or H; R₃ is

or H; R₄ is

or H; R⁵ is —CH₃, —C₂H₅, or —CH(CH₃)₂; X is an iodine or bromine atom;R^(5′) is a hydrogen or the group RCO, wherein R is hydrogen, branchedor unbranched alkyl of 1 to 10 carbon atoms, alkylene of 2 to 10 carbonatoms, aryl of 6 to 11 carbon atoms, a (C₆-C₁₁) aryl-alkyl (C₁-C₅)group, or a heteroaryl or heteroaryl-alkyl (C₁-C₅) group whereinheteroaryl is defined as 2- or 3-furyl, 2- or 3-thienyl, 2- or3-(N-methylpyrrolyl), 2-, 3-, or 4-pyridinyl, 2-, 4-, or5-(N-methylimidazolyl), 2-, 4-, or 5-oxazolyl, 2-, 3-, 5-, or6-pyrimidinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolyl, or 1-, 3-, 4-, 5-,6-, 7-, or 8-isoquinolyl, all aryl and heteroaryl groups optionallysubstituted by one or more hydroxy, amino, carboxy, halo, nitro, (C₁-C₃)alkoxy, or thioalkoxy of 1 to 5 carbon atoms; R₆ and R₇ are eachindependently selected from H and

R⁸ and R⁹ are each independently selected from hydrogen and straight andbranched-chain C₁-C₈ alkyl, wherein each said alkyl for R⁸ and R⁹ isindependently optionally substituted by —NH₂, —NHR¹¹, —NR¹¹R¹³, —OR¹¹,—OH, or —SR¹¹, wherein each R¹¹ and each R¹³ are independently selectedfrom straight and branched-chain C₁-C₅ alkyl; r is an integer 0 or 1; Gis oxygen or sulfur; Z¹ is H or straight or branched-chain C₁-C₅ alkyl;Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, twoor three groups independently selected from straight or branched-chainC₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴, —C(═O)NHR¹⁴,—O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴, —O(CH₂)_(n)C(═O)NHR¹⁴, and—S(CH₂)_(n)C(═O)NHR¹⁴, or Ar is a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-,1,8-, 2,3-, 2,6-, or 2,7-naphthylidene optionally substituted with one,two, three, or four groups independently selected from straight orbranched-chain C₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴,—C(═O)NHR¹⁴, —O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴,—O(CH₂)_(n)C(═O)NHR¹⁴, and —S(CH₂)_(n)C(═O)NHR¹⁴; wherein each R¹⁴ isindependently selected from (C₁-C₅)alkyl and each R¹⁴ is independentlyoptionally substituted with one or two groups selected from —OH,—(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl; each n is an integer independentlyselected from 0, 1, 2, 3, 4, and 5; W is selected from —O—, —S—,—C(═O)NH—, —NHC(═O)—, and —NR¹⁵—, wherein R¹⁵ is a (C₁-C₅)alkyl and R¹⁵is optionally substituted with one or two groups selected from —OH,—(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl; Y is a straight or branched-chain(C₁-C₆)alkylene group or a straight or branched-chain (C₁-C₆)alkenylenegroup; and Z is selected from the group consisting of

which method comprises a) treating a compound of Formula I

wherein R¹² is selected from straight and branched-chain C₁-C₈ alkyl,and each R¹⁰ is independently selected from hydrogen, R¹² and —OR¹²;with a strong acid to form a mixture comprising a compound of Formula IV

b) reacting the compound of Formula IV with a compound ZH; to form alinker intermediate of Formula V

and c) reacting the linker intermediate of Formula V resulting from step(b) with a methyltrisulfide compound CH₃—S—S—S-J; thereby synthesizing acalicheamicin derivative of Formula VI.
 108. A method according to claim107, wherein the compound of Formula I is obtained by reacting acompound of Formula II

with a compound of Formula III


109. A method according to claim 107, wherein one of R₆ and R₇ ishydrogen and the other of R₆ and R₇ is


110. A method according to claim 107, further comprising d) conjugatingthe calicheamicin derivative of Formula VI resulting from step (c) to amonoclonal antibody.
 111. A method according to claim 110, wherein themonoclonal antibody is inotuzumab or gemtuzumab.
 112. A method accordingto claim 107, wherein the methyltrisulfide compound CH₃—S—S—S-J isozogamicin.
 113. A method according to claim 112, further comprising d)conjugating the calicheamicin derivative of Formula VI resulting fromstep (c) to a monoclonal antibody.
 114. A method according to claim 113,wherein the monoclonal antibody is inotuzumab.
 115. A method accordingto claim 113, wherein the monoclonal antibody is gemtuzumab.
 116. Amethod of synthesizing a calicheamicin derivative of Formula VI

wherein J is

R₁ is

or CH₃; R₂ is

or H; R₃ is

or H; R₄ is

or H; R⁵ is —CH₃, —C₂H₅, or —CH(CH₃)₂; X is an iodine or bromine atom;R^(5′) is a hydrogen or the group RCO, wherein R is hydrogen, branchedor unbranched alkyl of 1 to 10 carbon atoms, alkylene of 2 to 10 carbonatoms, aryl of 6 to 11 carbon atoms, a (C₆-C₁₁) aryl-alkyl (C₁-C₅)group, or a heteroaryl or heteroaryl-alkyl (C₁-C₅) group whereinheteroaryl is defined as 2- or 3-furyl, 2- or 3-thienyl, 2- or3-(N-methylpyrrolyl), 2-, 3-, or 4-pyridinyl, 2-, 4-, or5-(N-methylimidazolyl), 2-, 4-, or 5-oxazolyl, 2-, 3-, 5-, or6-pyrimidinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolyl, or 1-, 3-, 4-, 5-,6-, 7-, or 8-isoquinolyl, all aryl and heteroaryl groups optionallysubstituted by one or more hydroxy, amino, carboxy, halo, nitro, (C₁-C₃)alkoxy, or thioalkoxy of 1 to 5 carbon atoms; R₆ and R₇ are eachindependently selected from H and

R⁸ and R⁹ are each independently selected from hydrogen and straight andbranched-chain C₁-C₈ alkyl, wherein each said alkyl for R⁸ and R⁹ isindependently optionally substituted by —NH₂, —NHR¹¹, —NR¹¹R¹³, —OR¹¹,—OH, or —SR¹¹, wherein each R¹¹ and each R¹³ are independently selectedfrom straight and branched-chain C₁-C₅ alkyl; r is an integer 0 or 1; Gis oxygen or sulfur; Z¹ is H or straight or branched-chain C₁-C₅ alkyl;Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, twoor three groups independently selected from straight or branched-chainC₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴, —C(═O)NHR¹⁴,—O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴, —O(CH₂)_(n)C(═O)NHR¹⁴, and—S(CH₂)_(n)C(═O)NHR¹⁴, or Ar is a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-,1,8-, 2,3-, 2,6-, or 2,7-naphthylidene optionally substituted with one,two, three, or four groups independently selected from straight orbranched-chain C₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴,—C(═O)NHR¹⁴, —O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴,—O(CH₂)_(n)C(═O)NHR¹⁴, and —S(CH₂)_(n)C(═O)NHR¹⁴; wherein each R¹⁴ isindependently selected from (C₁-C₅)alkyl and each R¹⁴ is independentlyoptionally substituted with one or two groups selected from —OH,—(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl; each n is an integer independentlyselected from 0, 1, 2, 3, 4, and 5; W is selected from —O—, —S—,—C(═O)NH—, —NHC(═O)—, and —NR¹⁵—, wherein R¹⁵ is a (C₁-C₅)alkyl and R¹⁵is optionally substituted with one or two groups selected from —OH,—(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl; Y is a straight or branched-chain(C₁-C₆)alkylene group or a straight or branched-chain (C₂-C₆)alkenylenegroup; and Z is selected from the group consisting of

which method comprises reacting a linker intermediate of Formula V

with a methyltrisulfide compound CH₃—S—S—S-J, in the presence of acarbodiimide; thereby synthesizing a calicheamicin derivative of FormulaVI.
 117. A method according to claim 116, wherein one of R₆ and R₇ ishydrogen and the other of R₆ and R₇ is


118. A method according to claim 116, wherein the carbodiimide is1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.
 119. A method accordingto claim 116, further comprising subjecting the calicheamicin derivativeof Formula VI to reversed phase purification protocol.
 120. The methodof claim 119, wherein the calicheamicin derivative of Formula VIresulting from the reversed phase purification protocol is subsequentlysubjected to a solid phase extraction protocol.
 121. A method accordingto claim 116, wherein the methyltrisulfide compound has an initialconcentration in said reaction of greater than about 3 g/L of thereaction mixture.
 122. A method according to claim 121, wherein themethyltrisulfide compound has an initial concentration in said reactionof between about 10 g/L and 110 g/L of the reaction mixture.
 123. Amethod according to claim 116, further comprising conjugating theresulting calicheamicin derivative of Formula VI to a monoclonalantibody.
 124. A method according to claim 123, wherein the monoclonalantibody is inotuzumab or gemtuzumab.
 125. A method according to claim116, wherein the methyltrisulfide compound CH₃—S—S—S-J is ozogamicin.126. A method according to claim 125, further comprising conjugating theresulting calicheamicin derivative of Formula VI to a monoclonalantibody.
 127. A method according to claim 126, wherein the monoclonalantibody is inotuzumab.
 128. A method according to claim 126, whereinthe monoclonal antibody is gemtuzumab.
 129. A method of purifying acalicheamicin derivative of Formula VI

wherein J is

R₁ is

or CH₃; R₂ is

or H; R₃ is

or H; R₄ is

or H; R⁵ is —CH₃, —C₂H₅, or —CH(CH₃)₂; X is an iodine or bromine atom;R^(5′) is a hydrogen or the group RCO, wherein R is hydrogen, branchedor unbranched alkyl of 1 to 10 carbon atoms, alkylene of 2 to 10 carbonatoms, aryl of 6 to 11 carbon atoms, a (C₆-C₁₁) aryl-alkyl (C₁-C₅)group, or a heteroaryl or heteroaryl-alkyl (C₁-C₅) group whereinheteroaryl is defined as 2- or 3-furyl, 2- or 3-thienyl, 2- or3-(N-methylpyrrolyl), 2-, 3-, or 4-pyridinyl, 2-, 4-, or5-(N-methylimidazolyl), 2-, 4-, or 5-oxazolyl, 2-, 3-, 5-, or6-pyrimidinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolyl, or 1-, 3-, 4-, 5-,6-, 7-, or 8-isoquinolyl, all aryl and heteroaryl groups optionallysubstituted by one or more hydroxy, amino, carboxy, halo, nitro, (C₁-C₃)alkoxy, or thioalkoxy of 1 to 5 carbon atoms; R₆ and R₇ are eachindependently selected from H and

R⁸ and R⁹ are each independently selected from hydrogen and straight andbranched-chain C₁-C₈ alkyl, wherein each said alkyl for R⁸ and R⁹ isindependently optionally substituted by —NH₂, —NHR¹¹, —NR¹¹R¹³, —OR¹¹,—OH, or —SR¹¹, wherein each R¹¹ and each R¹³ are independently selectedfrom straight and branched-chain C₁-C₅ alkyl; r is an integer 0 or 1; Gis oxygen or sulfur; Z¹ is H or straight or branched-chain C₁-C₅ alkyl;Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, twoor three groups independently selected from straight or branched-chainC₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴, —C(═O)NHR¹⁴,—O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴, —O(CH₂)_(n)C(═O)NHR¹⁴, and—S(CH₂)_(n)C(═O)NHR¹⁴, or Ar is a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-,1,8-, 2,3-, 2,6-, or 2,7-naphthylidene optionally substituted with one,two, three, or four groups independently selected from straight orbranched-chain C₁-C₆ alkyl, —OR¹⁴, —SR¹⁴, halogen, nitro, —COOR¹⁴,—C(═O)NHR¹⁴, —O(CH₂)_(n)COOR¹⁴, —S(CH₂)_(n)COOR¹⁴,—O(CH₂)_(n)C(═O)NHR¹⁴, and —S(CH₂)_(n)C(═O)NHR¹⁴; wherein each R¹⁴ isindependently selected from (C₁-C₅)alkyl and each R¹⁴ is independentlyoptionally substituted with one or two groups selected from —OH,—(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl; each n is an integer independentlyselected from 0, 1, 2, 3, 4, and 5; W is selected from —O—, —S—,—C(═O)NH—, —NHC(═O)—, and —NR¹⁵—, wherein R¹⁵ is a (C₁-C₅)alkyl and R¹⁵is optionally substituted with one or two groups selected from —OH,—(C₁-C₄)alkyl, and —S(C₁-C₄)alkyl; Y is a straight or branched-chain(C₁-C₆)alkylene group or a straight or branched-chain (C₂-C₆)alkenylenegroup; and Z is selected from the group consisting of

which method comprises subjecting the calicheamicin derivative ofFormula VI to a reversed phase high performance liquid chromatographypurification protocol.
 130. A method according to claim 129, wherein thereversed phase high performance liquid chromatography purificationprotocol comprises elution with phases comprising aqueous and organicmixtures, which phases range in pH from about 4 to about
 6. 131. Amethod according to claim 129, wherein one of R₆ and R₇ is hydrogen andthe other of R₆ and R₇ is


132. The method of claim 129, wherein the calicheamicin derivative ofFormula VI resulting from the reversed phase purification protocol issubsequently subjected to a solid phase extraction protocol.
 133. Amethod according to claim 129, wherein J is an ozogamicin moiety.
 134. Amethod according to claim 133, further comprising conjugating thecalicheamicin derivative of Formula VI so purified to a monoclonalantibody.
 135. A method according to claim 134, wherein the monoclonalantibody is inotuzumab.
 136. A method according to claim 134, whereinthe monoclonal antibody is gemtuzumab.
 137. A method of synthesizing acompound of Formula II

wherein R¹² is selected from straight and branched-chain C₁-C₈ alkyl;each R¹⁰ is independently selected from hydrogen, R¹² and —OR¹²; R⁸ andR⁹ are each independently selected from hydrogen and straight andbranched-chain C₁-C₈ alkyl, wherein each said alkyl for R⁸ and R⁹ isindependently optionally substituted by —NH₂, —NHR¹¹, —NR¹¹R¹³, —OR¹¹,—OH, or —SR¹¹, wherein each R¹¹ and each R¹³ are independently selectedfrom straight and branched-chain C₁-C₅ alkyl; r is an integer selectedfrom 0 and 1; and G is oxygen or sulfur; which method comprises treatinga compound of Formula VII

wherein R¹⁶ is —C(═O)OH or —C(═V)SH, wherein V is oxygen or sulfur, orR¹⁶ is —NH₂; with an azole activating agent of Formula IX

wherein V′ is oxygen or sulfur; and wherein E is

wherein m is an integer 0, 1, 2, or 3; q is an integer 0, 1 or 2; and pis an integer 0, 1, 2, 3, or 4; and wherein each R¹⁷ attached to E isindependently selected from straight and branched-chain (C₁-C₆)alkylgroups; in an organic solvent to form a compound of Formula VIII

wherein when R¹⁶ is —C(═O)OH, r is 0 and G is oxygen; when R¹⁶ is—C(═V)SH, r is 0 and G is V; and when R¹⁶ is —NH₂, r is 1 and G is V′;followed by combining the compound of Formula VIII with hydrazine,thereby forming a compound of Formula II.
 138. The method of claim 137,wherein the azole activating agent is selected from carbonyldiimidazole; thiocarbonyl diimidazole; carbonyl bis-pyrazole whereineach pyrazole optionally substituted with from one to three (C₁-C₆)alkyl groups; carbonyl bis-1,2,3-triazole; carbonyl bis-benzotriazole,and carbonyl bis-1,2,4-triazole.
 139. The method of claim 138, whereinthe azole activating agent is carbonyl diimidazole.
 140. The method ofclaim 137, wherein r is 0 and G is oxygen.
 141. The method of claim 137,wherein R¹⁶ in the compound of Formula VII is —C(═O)OH.
 142. The methodof claim 141, wherein the azole activating agent is carbonyldiimidazole.
 143. The method of claim 137, wherein the hydrazine isanhydrous hydrazine.
 144. The method of claim 137, wherein the hydrazineis hydrazine monohydrate.
 145. The method of claim 137, wherein thehydrazine is an aqueous solution of hydrazine.
 146. The method of claim137, wherein the hydrazine is a tetrahydrofuran solution of hydrazine.